Wendy Graham

Rapid detection of Parkinson's disease by SPECT with altropane: A selective ligand for dopamine transporters

Increasing evidence indicates that dopamine (DA) transporter density declines in Parkinsons disease (PD). 2β‐Carbomethoxy‐3β‐(4‐fluorophenyl)‐n‐(1‐iodoprop‐1‐en‐3‐yl) nortropane (IACFT, Altropane™) is a cocaine analog with high affinity and selectivity for dopamine transporter (DAT) sites in the striatum. In this study, single photon emission computed tomography (SPECT) with [123I]altropane was used to measure DAT density in seven healthy volunteers (five males, age 37–75, and two females, ages 26 and 39) and eight male patients with Parkinsons disease (age 14–79, Hoehn and Yahr stage: 1.5–3 (n = 5) and 4–5 (n = 3)). Dynamic SPECT images and arterial blood samples were acquired over 1.5–2 hr and plasma radioactivity was analyzed chromatographically to obtain metabolite corrected arterial input functions. Binding potential (BP, B′max/KD) for striatal (Str) DAT sites was calculated by two methods using occipital cortex (Occ) as a reference. In the first method, tissue time–activity curves (TAC) and metabolite corrected arterial input functions were analyzed by a linear graphical method developed for reversible receptor ligands. In the second method, the expression (StrTAC − OccTAC) was fitted to a gamma variate function and the maximum divided by OccTAC at the same time was used to estimate BP. In five of the PD patients, the SPECT data were compared with the results of PET with [18F] 6‐fluoro DOPA (FD‐PET). Plasma analysis indicated that [123I]altropane is rapidly converted to polar metabolites. SPECT images in healthy volunteers showed that [123I]altropane accumulated rapidly and selectively in the striatum and yielded excellent quality images within 1 h after injection. Both methods of analysis revealed a 7.6%/decade reduction in BP and average striatal values (corrected to age 25) were 1.83 ± 0.22 and 2.09 ± 0.20 by methods 1 and 2. In all the PD patients, striatal accumulation was markedly reduced and the pattern of loss was similar to that reported for DA; most profound in the posterior putamen with relative sparing of the caudate nuclei. A comparable pattern was observed with FD‐PET. For total striatum, age‐corrected BP was significantly (P < 0.001) reduced; 0.83 ± 0.06 (method 1), 0.84 ± 0.07 (method 2). BPs measured by the two methods were remarkably similar and highly correlated r2 = 0.88, (P < 0.001). These results indicate that [123I]altropane is an excellent SPECT ligand for imaging the DAT/DA neurons in human brain. The high selectivity and rapid striatal accumulation of the ligand allows for accurate quantitation of DAT sites in less than 2 hr. The results further demonstrate that [123I]altropane is an effective marker for PD. Synapse 29:128–141, 1998.

Comparison of the infection imaging properties of a 99mTc labeled chemotactic peptide with 111In IgG.

The biodistribution and infection imaging properties of a 99mTc labeled hydrazino nicotinamide (HYNIC) derivatized chemotactic peptide analog (For-Met-Leu-Phe-Lys-HYNIC) and 111In-DTPA-IgG were compared in rabbits with Escherichia coli infection. Six New Zealand white rabbits were injected in the left posterior thigh with a suspension of E. coli. Twenty four hours later, the animals were injected with: 1.0 mCi of 99mTc labeled peptide plus 0.1 mCi of 111In-DTPA-IgG. At 2-3 and 16-18 h, dual photon scintigrams were acquired and the images were corrected for crossover between the two windows. After recording the final images, the animals were sacrificed and biodistribution was determined. At both imaging times the biodistributions of the two reagents were markedly different. The highest concentrations of 111In-DTPA-IgG were detected in blood pool structures, liver and kidney. In contrast localization of 99mTc labeled peptide was greatest in spleen, lung and liver (consistent with binding to leukocytes). In general, the sites of infection were better visualized with the radiolabeled peptide and T/B ratios increased with time (P < 0.01). At both times, the T/Bs for 99mTc-peptide were higher (P < 0.01); 3.54 +/- 0.47 vs 2.52 +/- 0.38 at 2-3 h and 6.88 +/- 0.79 vs 3.78 +/- 0.36 at 16-18 h. These results indicate that although both radiopharmaceuticals localize at sites of infection, the radiolabeled peptide are superior reagents for the rapid detection of focal sites of infection. However, since the mechanisms of localization are different the combined use of both agents could have value in the general evaluation of infection/inflammation.

18F-labeling and biodistribution of the novel fluoro-quinolone antimicrobial agent, trovafloxacin (CP 99,219)

[18F]CP 99,219 [(1 alpha, 5 alpha, 6 alpha)-7-(6-amino-3-azabicyclo [3.1.0]hex-3-yl)-1-(2,4-difluorophenyl)-6-fluoro-1, 4-dihydro-4-oxo-1, 8-naphthyridine-3-carboxylic acid] was prepared by 18F for 19F exchange followed by reverse-phase HPLC purification. Studies of the effects of reaction time and temperature on 18F incorporation demonstrated that heating 1.0 mg of CP 99,219 in 0.5 cc of DMSO with 4.5 mg of K2CO3 and 24 mg of Kryptofix for 15 min at 160 degrees C results in the optimal compromise between radiochemical yield and purity. This method routinely provides radiochemical yields of 15-30% [EOS] with radiochemical purities of > 97%. Varying the concentration of CP 99,219 in the reaction mixture had no effect on yield. Biodistribution studies in rats demonstrated that significant concentrations of drug accumulate in most tissues. The tissues with the highest concentrations of drug were intestine, liver, kidney, and stomach.

Effect of adrenergic receptor ligands on metaiodobenzylguanidine uptake and storage in neuroblastoma cells

The effects of adrenergic receptor ligands on uptake and storage of the radiopharmaceutical [125I]metaiodobenzylguanidine (MIBG) were studied in the human neuroblastoma cell line SK-N-SH. For uptake studies, cells were incubated for 15 min with varying concentrations of α-agonist (clonidine, methoxamine, and xylazine), α-antagonist (phentolamine, tolazoline, phenoxybenzamine, yohimbine, and prazosin), β-antagonist (proranolol, atenolol), β-agonist (isoprenaline and salbutamol), mixed α/β antagonist (labetalol), or the neuronal blocking agent guanethidine, prior to the addition of [125I]MIBG (0.1 μM). The incubation was continued for 2 h and specific cell-associated radioactivity was measured. For the storage studies, cells were incubated with [125I]MIBG for 2 h, followed by replacement with fresh medium with or without drug (MIBG, clonidine, or yohimbine). Cell-associated radioactivity was measured at various times over the next 20 h. Propanolol reduced [125I]MIBG uptake by approximately 30% (P<0.01) at all concentrations tested, most likely due to nonspecific membrane changes. However, incubation with the other β-agonists or antagonists failed to elicit significant reductions in uptake. In contrast, all of the α-agonists significantly inhibited uptake (P<0.05); guanethidine >xylazine >clonidine=methoxamine. The α-antagonists demonstrated a broad range of inhibition (phenoxybenzamine ≫phentolamine ≫prazosin ≫yohimbine=tolazoline)(P<0.05). The mixed ligand, labetalol, inhibited MIBG uptake in a dose-dependent manner with an apparent IC50 of 0.65 μM. The retention studies demonstrated that unlabeled MIBG caused profound self-inhibition (P<0.01). Clonidine produced a modest inhibition of retention and yohimbine had no effect. Labetalol, phenoxybenzamine, guanethidine, and propranolol reduced uptake of [125I]MIBG by neuroblastoma cells in culture. Although only labetalol has been reported to cause false-negative MIBG scans, our results suggest that these other drugs have the potential to interfere with MIBG imaging and therapy, particularly at high doses. Adrenergic drugs did not alter cytoplasmic retention of [125I]MIBG in neuroblastoma cells but may have potential in tumors such as phenochromocytoma, where granular storage of MIBG has been observed. Inhibition of [125I]MIBG retention by unlabeled MIBG supports the use of high specific activity radioiodinated MIBG for both diagnosis and therapy.

Detection of acute bacterial infection within soft tissue injuries using a 99mTc-labeled chemotactic peptide

OBJECTIVE Infection imaging with a 99mTc-labeled chemotactic peptide was evaluated in a rabbit model of Escherichia coli infections in burned tissue. MATERIALS AND METHODS The peptide was radiolabeled with 99mTc via the hydrazino nicotinamide derivative. Three groups of six animals were studied: (group A) unilateral infected burns; (group B) bilateral burns with unilateral infection; and (group C) uninfected burns. Twenty-four hours after injury, groups A and B were infected, and 8 hours later, all animals were injected with approximately 0.50 mCi of 99mTc-peptide. MEASUREMENTS AND MAIN RESULTS In groups A and B, excellent images of the infections were obtained at 3 to 4 and 16 to 18 hours after injection of the peptide. At 3 to 4 hours after injection, the target-to-background ratios (T/B) were 3.12 +/- 0.28 for group A and 4.33 +/- 0.61 for group B (p = n.s.). At 16 to 18 hours, the T/B ratios increased significantly (p < 0.01): group A = 8.10 +/- 1.03 and B = 7.70 +/- 1.25. The T/B ratio for group C was only slightly greater than unity. CONCLUSIONS These results indicate that 99mTc-labeled chemotactic peptides are effective radiopharmaceuticals for the rapid detection of focal sites of infection within thermally injured tissues.

6-Mercaptomethylpyridine-3-carboxylic acid (MEMNIC): a new reagent for peptide labeling with Tc-99m

As part of our ongoing research into the development of peptide based radiopharmaceuticals, we have explored 2-mercaptopyridines and 2-mercapto-pyrimidines (2MPs) as coligands to control the radiochemical speciation of 99m Tc-labeled hydrazinonicotinamide (HYNIC) derivatized chemotactic peptides. In an attempt to develop an aminothiol ligand with greater stability, we synthesized 6-mercaptomethylpyridine–3-carboxylic acid (MEMNIC), assuming a five-member chelate ring complex would be more stable than the four-membered ring possible with 2MPs. Initial experiments suggested that 99m Tc–MEMNIC was stable in vivo and led to our investigation of it as a bifunctional chelator for 99m Tc labeling of peptides. The utility of MEMNIC was tested in a rabbit model of infection. The N-epsilon MEMNIC derivative of For–MLFK was prepared using the N-hydroxysuccinimidyl ester of MEMNIC. For–MLFK–MEMNIC was labeled via 99m Tc–mannitol and its imaging and biodistribution properties compared to Tc–For–MLFK–HYNIC and Tc–For–MLFCys (labeled via the free thiol of cysteine). Sites of infection were produced in New Zealand white rabbits (n=6/peptide) by injection of a suspension of Escherichia coli in a posterior thigh. Twenty-four hours after infection, the rabbits were injected intravenously with 0.5 mCi of HPLC purified 99m Tc-peptide and imaged at 2.5 and 18 h p.i. The animals were sacrificed at 18 h and tissue activity determined. At 2.5 and 18 h, the organ distribution of Tc–For–MLFK–HYNIC and Tc–For–MLFK–MEMNIC were qualitatively similar with the exception of higher renal accumulation of the latter, whereas Tc–For–MLFKCys showed rapid and prolonged accumulation in bowel. ROI analysis gave target/background ratios of 3.910.32 and 11.89 2.21 for Tc–For–MLFK–HYNIC, 5.09 0.66 and 9.881.12 for Tc–For–MLFK–MEMNIC and 2.590.16 and 1.70 0.37 for Tc–For–MLFCys, at 2.5 and 18 h. Tissue radioactivity measurements (18 h) demonstrated that compared to Tc–For–MLFK–HYNIC, the accumulation of Tc–For–MLFK–MEMNIC was less in all organs except in kidney which was greater. Direct labeling of the thiol group of cysteine led to lower accumulation in all organs compared to Tc–For–MLFK–MEMNIC, except in GI tract which was fivefold higher. While, Tc–For–MLFK–MEMNIC had half the accumulation of Tc–For–MLFK–HYNIC in infected muscle and pus, the lower accumulation in most organs and normal muscle resulted in good infection localization. These results indicate that MEMNIC can be successfully used to label peptides with 99m Tc while exhibiting good in vivo stability. The chemistry of MEMNIC with the {M(V)O} 3 + core characteristic of technetiun and its Group V congener rhenium was modeled by investigating the reactions of 2-mercaptomethylpyridine with appropriate Re(V)-oxo precursors in the presence of a variety of coligands. With diolate type tridentate donors of the class {X(CH2CH2O)2} 2 − (X= –NR, O, S), the ‘3+ 2’ compounds [ReO{ 3 -(OCH2CH2)2S}{ 2 SCH2C5H4N}] (2) and [ReO{ 3 -(OCH2CH2)2NCH3}{ 2 -SCH2C5H4N] (3) were isolated. In contrast, with the more sterically demanding dirmercapto class of tridentate ligands {S(CH2CH2S)2} 2 − ,t he ‘3+ 1’ species [ReO{ 3 -(SCH2CH2)2S}{ 1